TRANSLATIONAL REGULATION OF HEPATITIS-B VIRUS POLYMERASE GENE BY TERMINATION-REINITIATION OF AN UPSTREAM MINICISTRON IN A LENGTH-DEPENDENT MANNER

Hepatitis B virus (HBV) polymerase (P) gene is translated from the bic istronic pregenomic RNA with the core (C) gene in the first cistron, T he P ORF is preceded by the C AUG and three AUG codons within the C re gion, where a minicistron of 7 amino acids can potentially be translat ed. Our results indicate that the efficiency of the P gene translation initiation was about 10% of that of the C gene when both genes were f used in-frame to a lacZ reporter in an mRNA similar in structure to th e pregenomic RNA. By mutational analysis, about 74% of the translation initiation of HBV P gene was shown to be by ribosomes that reinitiate d after terminating translation of this minicistron, while the rest wa s by two mechanisms: one by ribosomes leaky scanning through every ups tream AUG and the other by ribosomal backwards scanning to the P AUG a fter finishing the translation of the C gene. The efficiency of termin ation-reinitiation depended on the size of the minicistron, i,e. the r einitiation efficiency decreased about 50% when the size increased fro m 24 nt to 57 nt, When a 44 nt HBV sequence comprising the minicistron was inserted at the 5' untranslated region of the cat gene, CAT expre ssion was regulated in a similar way to that of the HBV P gene. Moreov er, when transfection occurred with an HBV expression plasmid containi ng an inactivated minicistron, production of virus-like particles drop ped to about one-third of the wild-type level, suggesting that the ter mination-reinitiation mechanism is indeed important for HBV P gene exp ression.